Biology subjects

Doyle, D. A.

Publications and source records attributed to Doyle, D. A..

4 recordsLinked to original sources

bioRxiv2026

The iron-binding siderophore enterobactin is required for the response of multi-drug resistant Klebsiella pneumoniae to zinc limitation

To persist during infection Klebsiella pneumoniae must overcome nutrient iron and zinc limitation imposed by the host immune system through a process called nutritional immunity. Secreted small molecule siderophores are a major virulence determinant of Klebsiella pneumoniae pathogenesis and are presumed to overcome nutritional immunity by binding iron for bacterial acquisition. In this work, we set out to identify how a multi-drug resistant K. pneumoniae grows in zinc limited environments. Using unbiased transcriptomics, proteomics, and an arrayed transposon screen, we identified that synthesis and uptake of the siderophore enterobactin is required to allow for growth in low zinc conditions. Iron-specific chelators did not replicate this phenotype and addition of supplemental iron through heme in growth media could not complement severe growth defects of enterobactin mutant K. pneumoniae experiencing zinc limitation. Finally, zinc starvation induced enterobactin production independent of the canonical zinc uptake regulator (Zur) transcription factor suggesting an unidentified regulatory mechanism by which Gram-negative pathogens may respond to zinc stress. Together, these studies expand the role of enterobactin beyond iron regulation and highlight a previously unreported link between iron and zinc homeostasis in Klebsiella pneumoniae.

microbiology↗

bioRxiv2026

''Self-gapping'' by a C-terminal domain arginine finger regulates GTP hydrolysis in bacterial zinc metallochaperones

The cellular response to transition metal scarcity is multifaceted and complex. Members of the Cluster of Orthologous Groups 0523 (COG0523) superfamily are proposed to chaperone a bound metal to activate an apoenzyme client and are thus candidate metallochaperones. COG0523 enzymes are GTPases that harbor a conserved Ras-like GTP-binding and hydrolysis domain (G-domain) and a C-terminal domain (CTD) of unknown function connected by a flexible linker. AlphaFold3 modeling posits an "open" GTPase-inactive and "closed" GTPase-active conformation where the GTP and switch 1 (G2) loop are buried at the interface of the two domains. We show here that the CTD functions as a GTP-hydrolysis activation protein (GAP) domain that stimulates GTP hydrolysis by the tethered G-domain. This "self-gapping" activity requires an invariant RxKG sequence in the {beta}2-strand of the CTD in two distantly related bacterial COG0523s from Acinetobacter baumannii, ZigA and MigC. Thermodynamic and kinetic studies reveal that the Arg is analogous to the "arginine finger" motif of a Ras-cognate GAP, while the Lys residue appears to play a catalytic role in GTP hydrolysis. Cognate CTD added in trans to full-length RxK mutant ZigA or MigC rescues Zn(II)-activated GTPase activity whereas the non-cognate CTD shows no rescue. The linker in AbZigA appears to gate Zn(II)-stimulated GTP hydrolysis. Solution NMR studies of RxK AbMigC reveal that the two domains tumble independently of one another in the absence of bound ligands, with cognate CTD added in trans forming a tight complex. The extent to which conformational switching characterizes eukaryotic COG0523s is discussed. Significance StatementThe cellular response to nutrient transition metal limitation is evolutionarily conserved in all kingdoms, providing protection from the loss of these essential inorganic cofactors that power much of metabolism. An important part of this response is the increased cell abundance of members of the enigmatic and ubiquitous Cluster of Orthologous Groups 0523 (COG0523) superfamily. In bacterial pathogens, these enzymes are often associated with the low-zinc adaptive response to host-mediated nutritional immunity, where the host deploys transition metal chelation as an innate immune response to infections. In this work, we provide new mechanistic insights into COG0523 function with the discovery of "self-gapping" by the C-terminal domain of a two-domain G-protein architecture, placed into the context of a metallochaperone model.

biochemistry↗

bioRxiv2023

Evolutionary changes in germ granule mRNA content are driven by multiple mechanisms in Drosophila

The co-packaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to post-transcriptionally regulate mRNAs that function in germline development and maintenance. In D. melanogaster, mRNAs accumulate in germ granules by forming homotypic clusters, aggregates that contain multiple transcripts from a specific gene. Nucleated by Oskar (Osk), homotypic clusters in D. melanogaster are generated through a stochastic seeding and self-recruitment process that requires the 3 UTR of germ granule mRNAs. Interestingly, the 3 UTR belonging to germ granule mRNAs, such as nanos (nos), have considerable sequence variations among Drosophila species. Thus, we hypothesized that evolutionary changes in the 3 UTR influences germ granule development. To test our hypothesis, we investigated the homotypic clustering of nos and polar granule component (pgc) in four Drosophila species and concluded that homotypic clustering is a conserved developmental process used to enrich germ granule mRNAs. Additionally, we discovered that the number of transcripts found in nos and/or pgc clusters could vary significantly among species. By integrating biological data with computational modeling, we determined that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos, pgc, osk levels, and/or homotypic clustering efficacy. Finally, we found that the nos 3 UTR from different species can alter the efficacy of nos homotypic clustering, resulting in germ granules with reduced nos accumulation. Our findings highlight the impact that evolution has on the development of germ granules and may provide insight into processes that modify the content of other classes of biomolecular condensates.

evolutionary biology↗

bioRxiv2022

An In Vivo Analysis of the Functional Motifs of DEAD-box RNA Helicase Me31B in Drosophila Fertility and Germline Development

In Drosophila germline, Me31B is a putative ATP-dependent, RNA helicase that plays role in post-transcriptional RNA regulation to ensure the correct spatial and temporal expression of the mRNAs, a process crucial for proper germline development and fertility. However, Me31Bs in vivo working mechanism remains unclear. In this study, we aim to analyze the functions of Me31Bs key domains/motifs to understand how these domains/motifs operate to fulfill the proteins overall activities. We generated Drosophila strains mutant for six important motifs including three ATPase/helicase motifs (DEAD-box, DVLARAK, and HRIGR), the N-terminal domain (N-ter), the C-terminal domain (C-ter), and a protein-binding motif (FDF motif-binding motif). In characterizing these mutants, we observed that the three ATPase/helicase motif mutations cause dominant female sterility which is associated with developmental defects in oogenesis and embryogenesis. Follow-up examination of the DVLARAK motif mutant revealed its abnormalities in germline mRNA localization and transcript level. The Me31B N-ter domain (deletion of C-ter), C-ter domain (deletion of N-ter), and mutation of FDF motif-binding motif led to a decrease in female fertility and abnormal subcellular Me31B localizations in the egg chambers. Moreover, deletion of Me31B N-ter or C-ter motif results in a decrease of Me31B protein levels in the ovaries. This study indicates that these six motifs of Me31B play different roles to contribute to Me31Bs whole-protein functions like ATPase, RNA helicase, protein stability, protein localization, and partner protein binding, which are crucial for germline development and fertility. Considering Me31B protein familys conserved presence in both Drosophila germline and soma (for example, neurons) and in other organisms such as yeast, worm, mouse, and human, the results from this study could expand our understanding of Me31B helicase familys general working mechanisms in different cell types and species.

developmental biology↗